Photosynth Res
DOI 10.1007/s11120-006-9127-z
REGULAR PAPER
Quantification of cyclic electron flow around Photosystem
I in spinach leaves during photosynthetic induction
Da-Yong Fan Æ Qin Nie Æ Alexander B. Hope Æ
Warwick Hillier Æ Barry J. Pogson Æ Wah Soon Chow
Received: 31 August 2006 / Accepted: 11 December 2006
Springer Science+Business Media B.V. 2007
Abstract The variation of the rate of cyclic electron
transport around Photosystem I (PS I) during photosynthetic induction was investigated by illuminating
dark-adapted spinach leaf discs with red + far-red actinic light for a varied duration, followed by abruptly
turning off the light. The post-illumination re-reduction
kinetics of P700+, the oxidized form of the photoactive
chlorophyll of the reaction centre of PS I (normalized to
the total P700 content), was well described by the sum
of three negative exponential terms. The analysis gave a
light-induced total electron flux from which the linear
electron flux through PS II and PS I could be subtracted,
yielding a cyclic electron flux. Our results show that the
cyclic electron flux was small in the very early phase of
photosynthetic induction, rose to a maximum at about
30 s of illumination, and declined subsequently to
<10% of the total electron flux in the steady state.
Further, this cyclic electron flow, largely responsible for
the fast and intermediate exponential decays, was
sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethyl urea,
suggesting an important role of redox poising of the
cyclic components for optimal function. Significantly,
our results demonstrate that analysis of the post-illumination re-reduction kinetics of P700+ allows the
quantification of the cyclic electron flux in intact leaves
by a relatively straightforward method.
D.-Y. Fan
Laboratory of Quantitative Vegetation Ecology, Institute of
Botany, The Chinese Academy of Sciences, Beijing 100093,
China
Keywords Cyclic electron transport P700 Photosynthetic induction Photosystem I
Abbreviations
D.-Y. Fan Q. Nie W. Hillier W. S. Chow (&)
Photobioenergetics Group, Research School of Biological
Sciences, The Australian National University, Canberra,
ACT 0200, Australia
e-mail: [email protected]
ATP
Chl
DCMU
A. B. Hope
Faculty of Science and Engineering, School of Biological
Sciences, Flinders University, GPO Box 2100, Adelaide, SA
5001, Australia
Fd
Fv¢ and Fm¢
B. J. Pogson
School of Biochemistry and Molecular Biology, The
Australian National University, Canberra, ACT 0200,
Australia
MV
NADP+
Present Address:
Q. Nie
College of Resources and Planning Sciences, Jishou
University, Jishou, Hunan Province 427000, China
P700
PS I and PS II
PQ
Adenosine triphosphate
Chlorophyll
3-(3,4-Dichlorophenyl)-1,
1-dimethyl urea
Ferredoxin
Variable and maximum Chl
fluorescence during
illumination
Methyl viologen
Oxidized nicotinamide adenine
dinucleotide phosphate
Photoactive Chl of the reaction
centre of PS I
Photosystem I and II, respectively
Plastoquinone
123
Photosynth Res
Introduction
Arnon et al. (1955) demonstrated that ATP synthesis
is induced by illumination of thylakoids in the presence of vitamin K through a pathway of cyclic electron transport around Photosystem I (PS I). Since
then, great efforts have been made to understand the
physiological significance of this cyclic pathway.
Although current evidence supports an important role
for cyclic electron transport in C4 photosynthesis
(Herbert et al. 1990; Furbank et al. 1990), the evidence for and against cyclic electron transport as a
significant process in C3 plants (reviewed by Bendall
and Manasse 1995; Allen 2003; Buhkov and Carpentier 2004; Johnson 2005; Joliot and Joliot 2006) under
normal steady-state physiological conditions requires
further investigation. A strong piece of evidence for
an important role of cyclic electron transport in
steady-state conditions is in a recent report of an
Arabidopsis mutant that is incapable of cyclic electron
transport and, consequently, severely impaired in
growth and development (Munekage et al. 2004).
However, to our knowledge, quantification of the
cyclic electron flux in this mutant in comparison with
the wild type has yet to be made.
In terms of chloroplast energetics, if the current
view about the H+/e– ratio of 3 and the H+/ATP
ratio of 4.7 is correct (Allen 2003), only 2.55 ATP
molecules are synthesized per O2 molecule released,
which means an additional half an ATP molecule
needs to be generated via cyclic electron transport
for the fixation of each CO2 molecule, or via the
Mehler-ascorbate-peroxidase cycle. Furthermore, the
modulation of the ATP:NADPH ratio and the generation of a trans-thylakoid pH gradient via cyclic
electron transport may facilitate a flexible response
of leaves to changing environmental conditions
(Kramer et al. 2004). Thus, as demand for ATP increases relative to that for NADPH, cyclic electron
flow is increased, as supported by studies during the
photosynthetic induction period (Joliot and Joliot
2002, 2005, 2006), at elevated temperatures (Havaux
1996; Buhkov et al. 1999; Clarke and Johnson 2001),
at chilling temperatures (Clarke and Johnson 2001),
under anaerobiosis (Joët et al. 2002) and under
drought (Golding et al. 2004).
Primarily based on an analysis of the onset kinetics
of P700 photo-oxidation in dark-adapted leaves on
illumination with far-red light (Joliot and Joliot 2005,
2006), or the decay of 515 nm absorption during short
interruptions in illumination (Joliot and Joliot 2002), it
was concluded that there is a substantial initial rate of
123
cyclic electron transport on illumination of darkadapted leaves, and that pre-illumination induces a
transition from the cyclic to the linear mode. Measurements of the decay of oxidized P700 following brief
(up to 1 s) light flashes given to dark-adapted leaves
also support the transient cyclic electron flow in
previously dark-adapted leaves (Golding et al. 2004)
though no fluxes as such were presented. Similarly, the
changing of cyclic electron transport as a fraction of the
total during illumination is also indicated by a decrease
of Fv¢/Fm¢ (the quantum efficiency of open PS II reaction centres) from 0.76 at beginning to 0.64 after 5 min
far-red illumination at 1C (Cornic et al. 2000).
Unfortunately, quantification of the cyclic electron
flux during photosynthetic induction has not been
straightforward. For example, the method based on
the onset kinetics of P700 oxidation in dark-adapted
leaves is complicated by the extreme variability
(Joliot and Joliot 2005). Therefore, we sought an
alternative method that analyses the decay of the
P700+ signal after illumination (Maxwell and Biggins
1976), and from which the electron flux to P700+ can
be deduced (Chow and Hope 2004). To the extent
that we cannot separate charge recombination, if it
occurred, from energy-conserving cyclic electron flux
via the PQ pool and PS I, the cyclic electron flux that
we obtained may include a dissipative charge-recombination component.
In this paper, we evaluated the electron transport
flux to P700+ by fitting, with the sum of three negative
exponential components, the P700+ re-reduction
kinetics on cessation of red + far-red illumination. We
quantified the relative fraction of cyclic electron flow
during illumination of dark-adapted spinach leaves,
and showed that red + far-red illumination of darkadapted leaves gave rise to dynamic changes in cyclic
electron transport during photosynthetic induction. We
also investigated the relationship between cyclic electron flow and redox poise of electron carriers between
the two photosystems, showing that DCMU inhibited
not only the MV-resistant fast linear electron flux to
P700+, but also the MV-sensitive fast electron flux
which is attributable to cyclic electron flow.
Materials and methods
Spinacia oleracea L. cv. Yates hybrid 102 plants were
grown at 24/21C (day/night) with a 10-h photoperiod
(230 lmol photons m–2 s–1). The potting mixture was
supplemented by a slow release fertilizer (‘Osmocote’,
Scotts Australia Pty Ltd., Castle Hill).
Photosynth Res
Post-illumination re-reduction kinetics of P700+
Spectral Irradiance (µmol m-2 s-1 nm-1)
Redox changes of P700 in spinach leaf discs were
observed with a dual wavelength (820/870 nm) unit
(ED-P700DW) attached to a phase amplitude modulation fluorometer (Walz, Effeltrich, Germany) that was
used in the reflectance mode (Chow and Hope 2004). To
obtain the maximum signal corresponding to the total
amount of photo-oxidizable P700 (P700+max), a steadystate was first sought by illumination with far-red light
(12 lmol photons m–2 s–1, peak wavelength 723 nm,
102-FR, Walz, Effeltrich, Germany) for ‡10 s. Then a
single-turnover xenon flash (XST 103 xenon flash) was
applied to the adaxial side of the leaf disc. Flashes were
given at 0.1 Hz, and 16 consecutive signals were averaged (time constant = 95 ls). The maximum signal
amplitude immediately after the flash was taken as the
total amount of photo-oxidizable P700, and used to
normalize the signals obtained in the same geometry
with a stronger red + far-red actinic light (see below).
To measure the re-reduction kinetics of P700+ on
cessation of illumination with red + far-red actinic light,
leaf discs were first vacuum infiltrated with either
H2O, methyl viologen (MV, 300 lM) or DCMU
(100 lM, unless stated otherwise), blotted with absorbent paper, and allowed to evaporate off the excess
intercellular water, and then kept in a humidified container for a total of 1-h darkness before measurement.
Red + far-red actinic light from a slide projector was
selected by an RG 695 long-pass filter combined with a
Calflex C heat-reflection filter. The direction of the
actinic light made an angle of ca. 60 with the horizontal
leaf disc, while the light guide also made an angle of ca.
60 but was on the opposite side of the vertical. The
spectral irradiance measured by an LI1800 spectroradiometer (Licor, USA) is shown in Fig. 1, the irradiance
being ca. 260 lmol photons m–2 s–1 (675–750 nm).
Red + far-red actinic light was turned on by an electronic shutter triggered by a pulse/delay generator
(Model 555, Berkeley Nucleonics Corporation, USA)
for a required time interval and then turned off, while
another pulse from the signal generator triggered a
computer program to begin data acquisition (time constant = 2.3 ms) at 0.1 s before the actinic light was
turned off; data acquisition continued into the dark
period for 0.9 s. Signals from four to six separate leaf
discs were averaged. The duration of illumination given
to dark-adapted leaf discs was varied to investigate
variation of the electron fluxes to P700+ during
photosynthetic induction.
The longest possible duration of actinic illumination
controlled by the signal generator was 90 s. To extend
the duration of illumination, we repeatedly illuminated
8
6
4
2
0
600
650
700
750
800
Wavelength (nm)
Fig. 1 Spectral distribution of the red + far-red actinic light. The
red irradiance (£700 nm) was 70 lmol photons m–2 s–1, while the
far-red irradiance (701–750 nm) was 190 lmol photons m–2 s–1
leaf discs with bursts of actinic light, each of duration
2 s, and periodically stored the data for the post-illumination re-reduction kinetics of P700+. In this way we
were able to investigate cumulative periods of photosynthetic induction up to 10 min.
All post-illumination P700+ signals were normalized
to P700+max obtained in the same geometry (described
earlier) to give the fraction of oxidized P700 at any instant. During illumination, the concentration of P700+
was determined by the rate of photo-oxidation counteracted by re-reduction by electrons from various
sources. On abruptly turning off the actinic light, photooxidation ceased immediately, but the re-reduction
continued for some time. The initial rate of re-reduction
of P700+, therefore, should be equal to the electron flux
to P700+ immediately before cessation of illumination.
Hence, our objective was to determine the initial rate of
re-reduction of P700+ on abruptly turning off the
red + far-red actinic light. The re-reduction kinetics
(Fig. 2) were well fitted by the sum of three negative
exponentials, with normalized amplitudes A1, A2 and
A3, and rate coefficients k1, k2 and k3, yielding the total
initial rate of re-reduction of P700+ (hereafter termed
‘‘total electron flux’’) A1k1 + A2k2 + A2k2, in turnovers
of each P700 per second.
The rate of electron transport through PS II
measured by Chl a fluorescence
The relative Chl a fluorescence yield (F) at any instant
during photosynthetic induction and the maximum
123
Photosynth Res
A
1.0
P700+ / P700+max
0.8
2s+DCMU
0.6
2s+MV
0.4
0.2
2s+H2O
0.0
0.0
0.2
0.4
0.6
0.8
1.0
Time(s)
relative Chl a fluorescence yield at the same instant
(Fm¢) were measured. The Chl a fluorescence yield,
induced by blue modulated light, was measured at
wavelengths >660 nm, with little or no contamination
by PS I fluorescence. The average quantum yield of PS
II photochemistry, averaged over open and closed PS
II reaction centre traps, is given by 1 – F/Fm¢ (Genty
et al. 1989). This quantity can be used to calculate the
rate of electron transport through PS II (ETR, Schreiber 2004) as (1 – F/Fm¢) · I · A · f where I is the
irradiance, A the absorptance of the leaf and f is the
fraction of absorbed light partitioned to PS II. Since
ETR is directly proportional to (1 – F/Fm¢), we used
this quantity as a relative measure of the rate of linear
electron transport during photosynthetic induction.
Leaf discs infiltrated with H2O (control) or MV were
used after evaporation of excess intercellular water and
dark-adaptation for a total of 1 h.
Results
B
1.0
Post-illumination re-reduction kinetics of P700+ for
quantifying the electron flux to P700+
90s+DCMU
P700+ / P700+max
0.8
90s+MV
0.6
0.4
90s+H2O
0.2
0.0
0.0
0.2
0.4
0.6
0.8
1.0
Time(s)
Fig. 2 The re-reduction kinetics of P700+ in spinach leaf discs on
cessation of illumination with red + far-red actinic light. Red +
far-red light (ca. 260 lmol photons m–2 s–1) was turned on by an
electronic shutter for 2 s (A) or 90 s (B), then turned off at time
t = 0. The decay signal was recorded for 0.9 s. Each trace is the
average for four leaf discs. Leaf discs were cut from spinach
plants in a growth chamber during the photoperiod, infiltrated in
darkness with water, 100 lM DCMU or 300 lM MV (all with
0.5% ethanol, v/v) and allowed to evaporate off excess
intercellular water in darkness for 40 min. Afterwards, leaf discs
were put on wet filter paper for a further 20 min darkness (total
dark time = 60 min) before red + far-red light was given.
Further details are described in the ‘‘Materials and methods’’
section
123
Illumination of dark-adapted spinach leaf discs with
red + far-red actinic light led to a time-dependent extent
of net photo-oxidation of P700, while cessation of illumination resulted in re-reduction of P700+. In control
leaves infiltrated with water, 2 s red + far-red illumination gave a limited extent of photo-oxidation, which
rapidly relaxed in the post-illumination period
(Fig. 2A). On the other hand, the presence of either
DCMU (to inhibit PS II) or MV (to promote electron
flow from PS I to O2) resulted in 90% of P700 being
photo-oxidized even after just 2 s; re-reduction in
darkness was comparatively slow (Fig. 2A). At 90-s
illumination, >75% of the P700 was photo-oxidized even
in a control sample pre-infiltrated with H2O (Fig. 2B).
The re-reduction kinetics of P700+ were fitted as the
sum of three negative exponentials (slow, intermediate
and fast), yielding rate coefficients which, when multiplied by the respective amplitudes, gave the initial fluxes.
Both the rate coefficients and the fluxes are shown as a
function of duration of illumination of dark-adapted leaf
discs (Fig. 3). There was an order of magnitude difference between slow and intermediate, and between
intermediate and fast rate coefficients (Fig. 3A–C).
Similarly, successive initial fluxes also differ by an order
of magnitude (Fig. 3D–F). The total initial flux
(Fig. 3G) in H2O-infiltrated samples (solid circle),
equated with the total rate of electron flow to P700+ at
0.8
0.20
0.6
0.15
0.4
0.10
0.2
0.05
D
Slow
0.0
0.00
10
E
6
1.0
Flux (s-1)
1.5
8
4
0.5
2
Intermediate
B
0
Rate coefficient (s-1)
Flux (s-1)
0.25
0.0
50
15
40
30
10
20
Flux (s-1)
•
1.0
A
Rate coefficient (s-1)
Fig. 3 Rate coefficients (s–1)
and the fluxes (turnovers of
each P700 s–1) of slow,
intermediate and fast
components derived from the
sum of three negative
exponentials of best fit to the
post-illumination kinetics of
P700+ re-reduction. The
x-axis shows the time of
illumination (from 2 to 90 s)
before the actinic light was
turned off. Results are the
average of six experiments
using leaf discs infiltrated with
water ( ), 300 lM MV (s) or
100 lM DCMU (.). Total
flux = slow flux +
intermediate flux + fast flux.
Other conditions as for Fig. 2
Rate coefficient (s-1)
Photosynth Res
5
10
Fast
C
F
0
0
0
20
40
60
Time (s)
80
Total
10
Flux (s-1)
15
H2O
MV
DCMU
5
G
0
0
20
40
60
80
100
Time (s)
the instant the actinic light was turned off, increased with
time of illumination, reaching a peak at about 30 s of
illumination. In the presence of MV (open circles), the
total flux was relatively constant, while in the presence of
DCMU (inverted triangles), the flux declined to a small
value within 4 s of illumination (Fig. 3G).
Estimation of the linear electron flux
MV was expected to rapidly shunt electrons to oxygen
and abolish any cyclic electron flow, leaving only linear
electron flow. However, the difference in total fluxes
between H2O- and MV-infiltration cannot be directly
equated with the cyclic electron flux. This is because
photosynthetic induction manifested itself in H2Oinfiltrated samples but not in MV-infiltrated samples.
Therefore, to estimate the linear electron flux in the
presence of photosynthetic induction, we measured the
quantum yield of PS II photochemistry during illumination (1 – F/Fm¢), averaged over open and closed PS
II reaction centre traps (Genty et al. 1989). This
parameter is directly proportional to the rate of
123
Photosynth Res
A
20
15
c
Flux (s-1)
electron transport through PS II (Schreiber 2004).
Figure 4 shows the variation of (1 – F/Fm¢) during
photosynthetic induction, in leaves that had been
infiltrated with either H2O or MV. In the absence
of MV, there was a slight decrease in (1 – F/Fm¢) at
15–30 s of illumination, but an increase at 60–90 s.
Interestingly, (1 – F/Fm¢) was the same between MV
and H2O infiltration after 60–90 s of photosynthetic
induction. That is, the linear electron flux was the same
whether or not MV was present, provided sufficient
photosynthetic induction time had lapsed.
10
b
Estimation of the cyclic electron flux
5
We took advantage of this latter observation of the
same (1 – F/Fm¢) for MV- and H2O-infiltration after
60–90 s, so as to evaluate the cyclic electron flux during
photosynthetic induction. By scaling (1 – F/Fm¢) for
H2O-infiltrated samples in Fig. 4 to assume the same
flux value at 60 s as for MV-infiltrated samples in
Fig. 3G (open circles, middle curve), we could estimate
the linear flux, which is now plotted as curve (b) in
Fig. 5A. Subtracting curve (b) from curve (c) (total
flux, from the top curve of Fig. 3G) gave curve (a), the
estimated cyclic electron flux (Fig. 5A). Both cyclic
and linear electron fluxes are plotted in Fig. 5B, as a
percentage of their sum. It is clear that the linear
a
0
0
20
40
Time (s)
60
80
100
80
100
B
100
80
b
Flux (%)
1.0
60
40
0.8
1 - F/F'm
a
0.6
20
0.4
0
0
20
40
60
Time (s)
0.2
H2 O
MV
0.0
0
20
40
60
80
100
Time(s)
Fig. 4 Changes in the average quantum yield of PS II (1 –
F/Fm¢) as a function of photosynthetic induction time, when
dark-adapted spinach leaf discs were illuminated with red + farred actinic light. The dark time before each measurement was at
least 60 min. Each point is an average of five replicates (±SD)
123
Fig. 5 Cyclic electron flux (curve (a)) and linear electron flux
(curve (b)), as well as their sum (curve (c)), plotted as the
original value (A) or as a percentage of the total electron flux (B)
of dark-adapted spinach leaf discs subjected to illumination with
red + far-red light. Curve (c) in panel A was taken from the
H2O-infiltrated control in Fig. 3G (top curve). Curve (b) in panel
A was taken from the H2O-infiltrated control in Fig. 4, scaled to
assume the same flux value as the total flux for MV-infiltrated
samples at 60-s illumination in Fig. 3G (middle curve). The
rationale for this re-scaling is based on the observations that (i)
(1 – F/Fm¢)H2O = (1 – F/Fm¢)MV at 60 s (Fig. 4) and (ii) the rate
of linear electron transport through PS II is directly proportional
to (1 – F/Fm¢)
Photosynth Res
electron flux dominated at 2 s, decreasing rapidly to a
minimum at ca. 30 s and then recovering by 90 s. The
cyclic electron flux was small at 2 s, but peaked at
about 68% at 30 s, declining thereafter. At 90 s, about
37% of the total was cyclic electron flux.
Our existing signal generator did not allow us to
maintain the electronic shutter open for more than
90 s. To examine longer durations of illumination, we
resorted to cumulative illumination with 2-s flashes,
separated by 1.2 s dark time during which the postflash kinetics could be recorded if required. Figure 6
shows (in a log-scale for time) that H2O-infiltrated
samples exhibited a maximum total electron flux after
30-s cumulative illumination, but that the difference
between MV- and H2O-infiltration was very small
(~8%) after 600-s cumulative illumination.
The combined effect of DCMU and MV
on the total electron flux
The effect of DCMU on the cyclic electron flux
To investigate the action of DCMU on the electron
fluxes in detail, we varied the concentration of DCMU
in dark-adapted leaf discs that were all subjected
(without interruption) to 30-s illumination with
red + far-red light. This duration of illumination was
chosen to maximize the cyclic electron flux. Notably,
the greatest proportional decrease due to DCMU was
in the fast flux, regardless of the absence (solid circles)
20
H2O
MV
DCMU
15
Total Flux (s-1)
or presence (open circles) of MV; at 35 lM DCMU,
the fast flux was practically zero (Fig. 7F). The total
flux is shown in Fig. 7G. In the presence of MV, the
rate of linear electron flow was 20% greater than that
which occurred under conditions of photosynthetic
induction (30-s illumination, Fig. 4). Therefore, we
think that the true linear flux, allowing for an effect of
photosynthetic induction, is indicated by the dashed
line in Fig. 7G. The difference between the total
electron flux (solid line) and the true linear flux (dashed line) then gives the cyclic flux that was inhibited
by DCMU. Since the bulk of the total flux was contributed by the fast flux and was MV-sensitive, it implies that, at 30-s illumination, a substantial part of the
fast flux was indeed cyclic electron flow.
10
At 2-s illumination in the presence of DCMU, a small
flux of ca. 0.8 turnovers of each P700 s–1 could be observed (Fig. 3G). To elucidate the contributions to this
flux, we investigated the electron flux using DCMU
and MV separately or in combination, exposing darktreated leaves of another batch of plants to 2 s
red + far-red light (Fig. 8). In the presence of MV
alone, the flux was higher than that in H2O-infiltrated
samples, owing to the absence of photosynthetic
induction. Interestingly, in the combined presence of
DCMU and MV (i.e. in the absence of linear and cyclic
electron flow), the total electron flux was 0.77 s–1, as
compared with 0.84 s–1 in the presence of DCMU
alone. This residual electron flux of 0.77 s–1 at 2-s
illumination represents electron donation to the intersystem electron transport chain other than via linear or
cyclic electron flow. Its value decreased to 0.31 s–1 at
90-s illumination time (data not shown).
Discussion
5
The method of dark relaxation kinetics of P700+
on cessation of illumination
0
0
10
100
1000
Time (s)
Fig. 6 The average total electron flux (±SEM) from six
experiments using leaf discs infiltrated with water, 300 lM MV
or 100 lM DCMU as a function of cumulative time of
illumination with red + far-red actinic light (from 2 to 600 s).
Dark-adapted leaf discs were subjected to cycles of 2 s red + farred actinic light, 1.2 s darkness (the re-reduction kinetics of
P700+ were recorded during the 1.2 s darkness when required).
[Ethanol] = 0.5%
Our method analyses the post-illumination re-reduction kinetics of P700+ to obtain the rate of electron
flow during illumination immediately before cessation
of illumination. It is essentially equivalent to the
dark-interval relaxation kinetics (DIRK) analysis of
Sacksteder and Kramer (2000) in which the initial
rate of relaxation of a steady-state absorbance signal
is measured upon a light-to-dark transition. In our
application of this method, however, the leaves did
123
Photosynth Res
0.4
0.1
Flux (s-1)
0.2
0.6
0.2
A
D
0.0
0.0
Intermediate
Intermediate
8
1.2
6
0.8
4
0.4
2
Flux (s-1)
Rate coefficient (s-1)
•
0.3
Slow
Slow
0.8
Rate coefficient (s-1)
Fig. 7 The average rate
coefficients and the average
slow, intermediate and fast
fluxes as a function of DCMU
concentration (illumination
time = 30 s) from two
experiments using leaf discs
infiltrated with DCMU of
varied concentration either
with (s) or without ( )
300 lM MV. The decay of
P700+ was recorded on
cessation of illumination of
leaf discs with red + far-red
actinic light for 30 s. Other
conditions as for Fig. 2
0.0
E
B
0
20
16
40
12
30
8
20
4
10
Fast
C
Flux (s-1)
50
0
F
0
20
0
10
20
30
40
Total
16
DCMU concentration (µm)
12
8
H2 O
MV
Flux (s-1)
Rate coefficient (s-1)
Fast
4
0
G
0
10
20
30
DCMU concentration (µm)
not achieve a steady state before measurement,
because we were interested in the variation of electron fluxes during photosynthetic induction. Nevertheless, it appears valid to apply the method to a
non-steady-state situation since the rate of photooxidation of P700 and the rate of re-reduction of
P700+ together determine the net rate of change of
[P700+] at a given instant during illumination. Upon
turning off actinic light abruptly, photo-oxidation
ceases immediately, leaving re-reduction to occur
with an initial rate equal to that just prior to cessation of illumination. Golding et al. (2005) pointed
out that it should be possible to use the relaxation
kinetics of P700+ to estimate the electron flux
123
through the reaction centre, provided that P700 is
more than about 20% oxidized in the light. With this
constraint in mind, we would expect that our estimation of the total flux to P700+ to be valid for 4 s
photosynthetic induction time and longer (see below
for comments on estimates of total flux prior to 4 s).
Quantification of cyclic electron flow
The initial re-reduction kinetics of P700+ were
obtained by fitting the post-illumination relaxation
curves as the sum of three negative exponentials, from
which the initial slopes could be calculated to give the
Photosynth Res
8
Total flux (s-1)
6
4
2
0
H2O
DCMU
MV
DCMU
+MV
Fig. 8 The total electron flux to P700+ in leaves discs infiltrated
with H2O, DCMU, MV or DCMU + MV at photosynthetic
induction time 2 s. Leaves were dark-adapted for 1 h, and then
illuminated with red + far-red actinic light for 2 s before
measurement of the post-illumination kinetics of P700+. Other
conditions as for Fig. 3
total flux of electrons arriving at P700+ immediately
before cessation of illumination. The total electron
flux, increasing to a maximum at 30 s induction time
and decreasing thereafter in H2O-infiltrated leaf samples, was much diminished in the presence of MV
which abolished cyclic electron flow (Fig. 3G). However, the total linear electron flux in the presence of
MV did not undergo any significant induction, whereas
the total flux in H2O-infiltrated samples did. Therefore,
one cannot simply use the difference between the two
fluxes to estimate the cyclic electron flux while photosynthetic induction was in progress. To derive the
linear electron flux during photosynthetic induction,
we monitored the quantity (1 – F/Fm¢) which is directly
proportional to the linear electron flow through PS II.
Interestingly, such a relative measure of linear electron
flow through PS II was identical for H2O- and MVinfiltrated leaf discs at 60 s of photosynthetic induction
or longer (Fig. 4). This coincidence allowed us to scale
the relative linear flux in H2O-infiltrated leaf discs in
Fig. 4 (open circles) so that, at 60 s, it has the same
numerical value as for MV-infiltrated samples in
Fig. 3G (open circles), giving curve (b) in Fig. 5A.
Then it was possible to obtain the cyclic electron flux
(curve (a)) as the difference between the total flux
(curve (c)) and the linear flux (curve (b)) during photosynthetic induction in Fig. 5A. The linear electron
flux through PS II could consist of electron flow to
NADP+ or to O2 via a Mehler-ascorbate-peroxidase
reaction. Both partial linear electron fluxes might undergo induction, but they both occurred in curves (b)
and (c) in Fig. 5A, so subtraction cancels both linear
partial fluxes, allowing quantification of the cyclic
electron flux.
Figure 5B curve (a) shows that the cyclic flux, as a
fraction of the total, increased to a maximum of about
68% at 30 s. At the same time the contribution of
linear electron flow fell to a minimum, presumably due
to its down-regulation by the pH gradient generated by
cyclic electron flow (Heber and Walker 1992) and
possibly by O2-dependent electron flow (Schreiber
et al. 1995). Subsequently, the cyclic flux declined to
about 37% at 90 s.
At long photosynthetic induction times (several
minutes), the cyclic electron flux, in our leaf discs under moderately low red + far-red actinic irradiance,
declined to a few percent of the total (Fig. 6B). This
agrees with the observations of Joliot and Joliot (2006)
for moderate green light and those of Laisk et al.
(2005). Given the hypothesis that the relative occurrence of linear and cyclic electron flow depends on the
rate of electron transfer from Fd- to NADP+ via ferredoxin-NADP reductase (Joliot and Joliot 2006), we
expect that during steady-state photosynthesis under
normal conditions, NADP+ is relatively abundant, and
readily accepts electrons from Fd–. Under such conditions, linear electron flow out-competes cyclic flow. On
the other hand, in anaerobic (Joët et al. 2002), chilling
(Clarke and Johnson 2001) or drought conditions
(Golding et al. 2004), carbon assimilation is retarded,
cyclic electron flow is increased, and linear electron
flow is diminished, as expected.
The cyclic electron flux during early photosynthetic
induction was low
On a time scale of minutes, the data of Joliot and Joliot
(2006), obtained from the onset phase of P700 photooxidation, revealed that the cyclic flux constitutes
about 85% of the total at the earliest time point (about
5 s). In contrast, our earliest time point at 2 s yielded a
small cyclic flux (ca. 12% of the total electron flux,
Fig. 5 curve (b)). However, since our cyclic flux
increased threefold by 4 s, there may not be any serious discrepancy at 4–5 s between the two observations,
which were made under different light conditions.
Even our slow cyclic flux at 2 s was almost certainly
an overestimate. The total flux to P700+ strictly consists
of three partial fluxes: a cyclic flux, a PS II linear
electron flux and a stromal flux that originated in
123
Photosynth Res
stromal reductants feeding electrons into the PQ pool.
Simple subtraction of the PS II linear electron flux
from the total, therefore, overestimated the cyclic flux,
particularly before stromal reductants were depleted
by light-induced electron transfer to NADP+ or O2.
Indeed, as seen in Fig. 8 in the combined presence of
DCMU (an inhibitor of linear electron flow) and MV
(a competitor of cyclic electron flow), the residual
electron flux of 0.77 turnovers of each P700 s–1 (~13%
of the total electron flux) at 2 s was almost certainly
due to electron flow from stromal reductants. This
stromal flux declined to 0.31 s–1 (~3% of the total
electron flux) by 90-s illumination (data not shown),
presumably due to partial depletion of the pool of
stromal reductants.
Poising of cyclic electron flow by electrons
from PS II
The fast electron flux with a rate coefficient of 30–50 s–1
(depending on the presence or absence of MV), dominated the post-illumination kinetics of P700+ after 30 s
actinic light. Its marked sensitivity to MV (Fig. 7F)
strongly suggests that it is a cyclic electron flux. Further, this MV-sensitive cyclic electron flow was sensitive to DCMU, which blocks linear electron transfer in
PS II. We interpret the DCMU effect on the MVsensitive component as being due to inhibition of the
redox poising of the intersystem electron transport
chain. Cyclic electron flow cannot occur if its components are either completely reduced (because there is
nowhere for the electrons to go) or completely oxidized (because there are no electrons to cycle)
(Whatley 1995; Allen 2003). The red + far-red actinic
light used in this study had a component with wavelengths below 700 nm capable of stimulating PS II
(Chow and Hope 2004; Hughes et al. 2006); electrons
from water oxidation in PS II could then be injected
into the cyclic chain to poise it for optimal cyclic flow.
In the presence of >30 lM DCMU, however, such
poising was prevented, resulting in the loss of not only
(1) the MV-resistant fast flux (attributable to linear
electron transport from PS II to P700+), but also (2) the
MV-sensitive fast flux (attributable to cyclic electron
flow) (Fig. 7F).
In this study, we have not addressed the various
possible routes of cyclic electron flow around PS I,
mediated by (1) the putative ferredoxin-plastoquinone
reductase (Bendall and Manasse 1995) as regulated by
the pgr5 gene product (Munekage et al. 2004) and (2)
NAD(P)H dehydrogenase (Mi et al. 1995) or the
ferredoxin-NADP reductase (Buhkov and Carpentier
2004). Nor have we addressed the possibility that
123
the different post-illumination relaxation phases
correspond to (1) populations of PS I with distinct
properties (Albertsson 1995), (2) the same enzymes
located in different membrane domains (Buhkov et al.
2002), (3) different enzymes mediating different routes
(Chow and Hope 2004), or (4) heterogeneity of PS II
reaction centres. Instead, we quantified, by subtraction
of the linear electron flux from the total flux, a cyclic
electron flux which might represent the combined
contributions of different routes.
In conclusion, our results show that during photosynthetic induction under moderately low red + farred light, the rate of cyclic electron transport started
from a low value, increased to a maximum of 11–15
turnovers of each P700 s–1 (68% of the total electron
flux) after about 30 s of illumination, and declined
gradually until it was <10% of the total electron flux
through PS I in the steady state. Further, we have
demonstrated the quantification of the cyclic electron
flux with a relatively straightforward method.
Acknowledgements DF is supported in part by the National
Natural Science Foundation of China (No. 90302004) and
Knowledge Innovation Program of the Chinese Academy of
Sciences (KSCX2-SW-109). Partial financial support of this
project from an ARC grant DP 0665363 to BJP and WSC is
gratefully acknowledged. ABH is grateful for a Visiting Fellowship from RSBS, ANU. We thank Jan Anderson and Tom
Wydrzynski for constructive comments on the manuscript.
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